Entry into mitosis catalyzed by the Cdc2/Cyclin B kinase, which phosphorylates multiple proteins to induce the dramatic cellular rearrangements characteristic of mitosis. When DNA replication is inhibited, a cell cycle checkpoint pathway prevents mitosis through inhibitory phosphorylation of Cdc2 at Y15 and T14. This inhibition involves sustained activity of the Cdc2 Y15-directed kinase, Wee1, as well as suppression of the Cdc2 Y15 phosphatase, Cdc25. We have discovered that a novel G2/M regulator, Hsl7, promotes intranuclear Wee1 degradation, and that the DNA replication checkpoint disrupts Hsl7-Wee1 interactions. In parallel, Cdc25 is inhibited through binding to 14-3-3 protein. Removal of 14-3-3 from Cdc25 is required for its mitotic activation, and we have found that dissociation of the 14-3-3-Cdc25 complex requires phosphorylation of both Cdc25 T138 (which reduces the affinity of 14-3-3 for Cdc25) and intermediate filament proteins (which then bind 14-3-3, thereby serving as an abundant 14-3-3 "sink"). Additionally, we have discovered that a previously described apoptotic inhibitor, Aven, can also regulate mitotic entry and may be important for DNA-responsive checkpoint operation. The objective of this proposal to elucidate both the Wee1 and Cdc25-modulatory arms of DNA-responsive checkpoint pathways with the long term goal of fully understanding the control of M phase entry. Towards this end, we propose to I) Delineate the role of Hsl7 in checkpoint-mediated Wee1 regulation II) Elucidate the mechanism(s) which regulate 14-3-3 release from Cdc25 and III) Determine how Aven regulates mitotic entry.